Many regulatory proteins are rapidly degraded. In addition, the classic response to starvation, nutrient deprivation and/or stress includes an increased rate of intracellular proteolysis. At least three general types of proteolytic reactions have been implicated: proteolysis by soluble or membrane-bound proteases (often responding to second signals such as calcium); up-regulation of the lysosomal proteolytic machinery upon carbon starvation; and stimulation of the ubiquitin-dependent proteolysis system. The focus of this grant is the latter system. Ubiquitin has been shown to be involved in the regulated proteolysis of damaged proteins, as well as short-lived regulatory molecules such as cyclins, c-mos, p53, c-myc, c- fos, c-jun, and NF-kB. This system also has been shown to respond to glucocorticoids during fasting, to TNF as may occur in cachexia, to metabolic acidosis, to interferon gamma elicited by viral infection, to feeding cycles, to heat-shock, and to damaged proteins. It is apparent that cellular proteolysis is regulated in response to intracellular signals, many of which are ill-defined. Both the lysosomal and the ubiquitin-dependent systems function as compartmentalized multienzyme systems which sequester proteolytic sites from the bulk of soluble proteins. The lysosome packages its proteases in an acidic compartment and acquires substrates by either autophagy or selective uptake through the hsp73 receptor. The ubiquitin-dependent system sequesters its proteolytic sites by inclusion in a multi-enzyme complex and selects substrates by ubiquitinylation of the target protein. This post-translational covalent modification commits the target proteins to degradation. As such, the enzymes which reverse this modification (the UCH and UBP proteins) are also important in regulating flux through this system. This proposal will characterize the structure, function and substrate specificity of the UCH/UBP families of enzymes. The long-term goals are to elucidate this system of regulation and describe how it functions in normal metabolism, in the stress response, and in the control of cell- cycle events. The results will have broad implications for understanding oncogenes, carcinogenesis, receptor function, and pathological protein turnover such as inclusion body formation or cachexia. The specific aims are: 1) The mechanism and specificity of UCH (Ubiquitin C-terminal Hydrolase) isozymes will be defined, 2) The proteasome-associated isopeptidase activity will be purified and characterized 3) The structure mechanism and specificity of UBP isozymes (Ubiquitin Specific Proteases) will be examined.